Use of green porphyrins to treat neovasculature in the eye

ABSTRACT

Photodynamic therapy of conditions of the eye characterized by unwanted neovasculature, such as age-related macular degeneration, is effective using green porphyrins as photoactive agents, preferably as liposomal compositions.

This application is a continuation of application U.S. Ser. No.10/982,409, filed on Nov. 5, 2004, which is a continuation ofapplication U.S. Ser. No. 10/418,965, filed on Apr. 18, 2003, which is acontinuation of application U.S. Ser. No. 09/824,155, filed on Apr. 2,2001, now U.S. Pat. No. 6,610,679, which is a continuation of U.S. Ser.No. 09/347,382, filed on Jul. 6, 1999, now U.S. Pat. No. 6,225,303,which is a continuation of U.S. Ser. No. 08/942,475, filed Oct. 2, 1997,now abandoned, which is a continuation of U.S. Ser. No. 08/390,591,filed Feb. 17, 1995, now U.S. Pat. No. 5,798,349, which is acontinuation-in-part of U.S. Ser. No. 08/209,473, filed Mar. 14, 1994,now U.S. Pat. No. 5,707,986; the entire contents of each of theseapplications are hereby incorporated by reference.

TECHNICAL FIELD

The invention is in the field of photodynamic therapy, specificallyrelated to ocular conditions. More particularly, the invention concernsthe use of green porphyrins in photodynainic therapeutic treatment ofconditions characterized by unwanted neovasculature in the eye.

BACKGROUND ART

Choroidal neovascularization leads to hemorrhage and fibrosis, withresultant visual loss in a number of eye diseases, including maculardegeneration, ocular histoplasmosis syndrome, myopia, and inflammatorydiseases. Age-related macular degeneration (AMD) is the leading cause ofnew blindness in the elderly, and choroidal neovascularization isresponsible for 80% of the severe visual loss in patients with thisdiseases. Although the natural history of the disease is eventualquiescence and regression of the neovascularization process, thisusually occurs at the cost of sub-retinal fibrosis and vision loss.

Current treatment of AMD relies on occlusion of the blood vessels usinglaser photocoagulation. However, such treatment requires thermaldestruction of the neovascular tissue, and is accompanied byfull-thickness retinal damage, as well as damage to medium and largechoroidal vessels. Further, the subject is left with an atrophic scarand visual scotoma. Moreover, recurrences are common, and visualprognosis is poor.

Developing strategies have sought more selective closure of the bloodvessels to preserve the overlying neurosensory retina. One such strategyis photodynamic therapy, which relies on low intensity light exposure ofphotosensitized tissues to produce photochemical effects.Photosensitizing dyes are preferentially retained in tumors andneovascular tissue, which allows for selective treatment of thepathologic tissue. As a result of the invention, PDT may be used tocause vascular occlusion in tumors by damaging endothelial cells, aswell as a direct cytotoxic effect on tumor cells.

Photodynamic therapy of conditions in the eye characterized byneovascularization has been attempted over the past several decadesusing the conventional porphyrin derivatives such as hematoporphyrinderivative and Photofrin porfimer sodium. Problems have been encounteredin this context due to interference from eye pigments. In addition,phthalocyanine has been used in photodynamic treatment.

A newer photosensitizer, a member of the group designated “greenporphyrins”, is in the class of compounds called benzoporphyrinderivatives (BPD). This photosensitizer has also been tested to someextent in connection with ocular conditions. For example, Schmidt, U. etal. described experiments using BPD coupled with low density lipoprotein(LDL) for the treatment of Greene melanoma (a nonpigmented tumor)implanted into rabbit eyes and achieved necrosis in this context (IOVS(1992) 33:1253 Abstract 2802). This abstract also describes the successof LDL-BPD in achieving thrombosis in a corneal neovascularizationmodel. The comeal tissue is distinct from that of the retina andchoroid.

The present applicants have described treating choroidalneovascularization using LDL-BPD in several abstracts published 15 Mar.1993. These abstracts include those by Schmidt-Erfurth, U. et a].(abstract 2956); by Haimovici, R. et al. (abstract 2955); and by Walsh,A. W. et al. (abstract 2954). In addition, Lin, S. C. et al. describedphotodynamic closure of choroidal vessels using liposomal BPD in(abstract 2953). All of the foregoing are published in IOVS (1993)34:1303. An additional abstract of the present applicants describingLDL-BPD to inhibit choroidal neovasculature is by Moulton, R. S. et al.(abstract 2294), IOVS (1993) 34: 1169.

The green porphyrins offer advantages in their selectivity forneovasculature. The present applicants have further determined thatcoupling of the green porphyrins to a carrier such as LDL or ascontained in a liposomal formulation provides an advantageous deliverymethod for the drug to the desired ocular location.

Disclosure of the Invention

The invention is directed to diagnosis and treatment of certainconditions of the eye using photodynamic methods and employing greenporphyrins as the photoactive compounds. The green porphyrins of theinvention are described in U.S. Pat. Nos. 4,883,790; 4,920,143;5,095,030; and 5,171,749, the entire contents of which are incorporatedherein by reference. These materials offer advantages of selectivity andeffectiveness when employed in protocols directed to the destruction ofunwanted ocular neovasculature, especially in the choroid.

Accordingly, in one aspect, the invention is directed to a method totreat conditions of the eye characterized by unwanted neovasculature,which method comprises administering to a subject in need of suchtreatment an amount of a liposomal formulation of green-porphyrin thatwill localize in said neovasculature; and irradiating the neovasculaturewith light absorbed by the green porphyrin.

In another aspect, the invention is directed to a method to treatconditions of the choroid characterized by unwanted neovascularization,such as AMD, which method comprises administering to a subject in needof such treatment an amount of a green porphyrin that will localize inthe neovascularized choroid; and irradiating the choroid with lightabsorbed by the green porphyrin.

In still another aspect, the invention is directed to a method to treatage-related macular degeneration (AMD) which method comprisesadministering to a subject in need of such treatment an amount of greenporphyrin that will localize in the choroid and irradiating the choroidwith light absorbed by the green porphyrin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows preferred forms of the green porphyrins useful in themethods of the invention.

MODES OF CARRYING OUT THE INVENTION

In general, the green porphyrin is of a formula shown in FIG. 1 or amixture thereof.

Referring to FIG. 1, in preferred embodiments each of R¹ and R² isindependently selected from the group consisting of carbalkoxyl (2-6C),alkyl (1-6C), arylsulfonyl (6-10C), cyano and —CONR⁵CO wherein R⁵ isaryl (6-10C) or alkyl (1-6C); each R³ is independently carboxyl,carboxyalkyl (2-6C) or a salt, amide, ester or acylhydrazone thereof oris alkyl (1-6C); R⁴ is —CH═CH₂ or —CCH(OR⁴′) CH3 wherein R⁴′ is H, oralkyl (1-6C) optionally substituted with a hydrophilic substituent.Especially preferred also are green porphyrins of the formula shown inFIGS. 1-3 or 1-4 or mixtures thereof.

More preferred embodiments are those wherein the green porphyrin is ofthe formula shown in FIG. 1-3 or 1-4 or a mixture thereof and whereineach of R¹ and R² is independently carbalkoxyl (2-6C); one R³ iscarboxyalkyl (2-6C) and the other R³ is an ester of a carboxyalkyl(2-6C) substituent; and R⁴ is —CCH═CH₂ or —CCH(OH)CH₃.

Still more preferred are embodiments wherein green porphyrin is of theformula shown in FIG. 1-3 and wherein R¹ and R² are methoxycarbonyl; oneR³ is —CCH₂CH₂COOCH₃ and the other R³is —CCH₂CH₂COOH; and R⁴ is—CCH═CH₂; i.e., BPD-MA.

The green porphyrin is formulated into a delivery system that delivershigh concentrations to the target tissue. Such formulations may includecoupling to a specific binding ligand which may bind to a specificsurface component of the neovasculature or by formulation with a carrierthat delivers higher concentrations to the target tissue.

In one preferred embodiment, the green porphyrin is prepared as aliposomal formulation. Liposomal formulations are believed to deliverthe green porphyrin selectively to the low-density lipoprotein componentof plasma which, in turn acts as a carrier to deliver the activeingredient more effectively to the neovasculature. Increased numbers ofLDL receptors have been shown to be associated with neovascularization,and by increasing the partitioning of the green porphyrin into thelipoprotein phase of the blood, it appears to be delivered moreefficiently to the neovasculature. Green porphyrins, and in particularBPD-MA, strongly interact with such lipoproteins. LDL itself can be usedas a carrier, but LDL is considerably more expensive and less practicalthan a liposomal formulation. LDL, or preferably liposomes, are thuspreferred carriers for the green porphyrins since green porphyrinsstrongly interact with lipoproteins and are easily packaged inliposomes. Compositions of green porphyrins involving lipocomplexes,including liposomes, are described in U.S. Pat. No. 5,214,036 and inU.S. Ser. No. 07/832,542 filed 5 Feb. 1992, the disclosures of both ofthese being incorporated herein by reference. Liposomal BPD can also beobtained from Quadra Logic Technologies, Inc., Vancouver, BritishColumbia.

When injected intravenously, BPD-MA is cleared from the bloodstream witha half-life of about 10-30 minutes, with the highest tissue levels beingreached in about three hours after administration by injection anddeclining rapidly in the first 24 hours. BPD-MA is cleared primarily viabile and feces (60%), with only 4% being cleared via the kidneys andurine. Thus, skin photosensitivity occurs with BPD-MA only transiently,with minimal reactivity after 24 hours in in vivo models.

The green porphyrin can be administered in any of a wide variety ofways, for example, orally, parenterally, or rectally. Parenteraladministration, such as intravenous, intramuscular, or subcutaneous, ispreferred. Intravenous injection is especially preferred.

The dose of green porphyrin can vary widely depending on the tissue tobe treated; the physical delivery system in which it is carried, such asin the form of liposomes; or whether it is coupled to a target-specificligand, such as an antibody or an immunologically active fragment.

It should be noted that the various parameters used for effective,selective photodynamic therapy in the invention are interrelated.Therefore, the dose should also be adjusted with respect to otherparameters, for example, fluence, irradiance, duration of the light usedin photodynamic therapy, and time interval between administration of thedose and the therapeutic irradiation. All of these parameters should beadjusted to produce significant damage to neovascular tissue withoutsignificant damage to the surrounding tissue. Typically, the dose ofgreen porphyrin used is within the range of from about 0.1 to about 20mg/kg, preferably from about 0.15-2.0 mg/kg, and even more preferablyfrom about 0.25 to about 0.75 mg/kg.

Specifically, as the green porphyrin dose is reduced from about 2 toabout 1 mg/kg, the fluence required to close choroidal neovasculartissue tends to increase, for example, from about 50 to about 100Joules/cm².

After the photosensitizing green porphyrin has been administered, theneovascular tissue or tumor being treated in the eye is irradiated atthe wavelength of maximum absorbance of the green porphyrin, usuallybetween about 550 and 695 nm. A wavelength in this range is especiallypreferred for enhanced penetration into bodily tissues.

As a result of being irradiated, the green porphyrin in its tripletstate is thought to interact with oxygen and other compounds to formreactive intermediates, such as singlet oxygen, which can causedisruption of cellular structures. Possible cellular targets include thecell membrane, mitochondria, lysosomal membranes, and the nucleus.Evidence from tumor and neovascular models indicates that occlusion ofthe vasculature is a major mechanism of photodynamic therapy, whichoccurs by damage to endothelial cells, with subsequent plateletadhesion, degranulation, and thrombus formation.

The fluence during the irradiating treatment can vary widely, dependingon type of tissue, depth of target tissue, and the amount of overlyingfluid or blood, but preferably varies from about 50-200 Joules/cm².

The ikradiance typically varies from about 150-900-mW/cm², with therange between about 150-600 mW/cm² being preferred. However, the use ofhigher irradiances may be selected as effective and having the advantageof shortening treatment times.

The optimum time following green porphyrin administration until lighttreatment can vary widely depending on the mode of administration, theform of administration such as in the form of liposomes or as a complexwith LDL, and the type of target tissue. As a specific example, anexposure time of 1-20 minutes is often appropriate for retinalneovascular tissue, about 120 minutes for choroidal neovascular tissue,and up to about three hours for tumors. Thus, effective vascular closuregenerally occurs at times in the range of about one minute to aboutthree hours following administration of the green porphyrin.

The time of light irradiation after administration of the greenporphyrin may be important as one way of maximizing the selectivity ofthe treatment, thus minimizing damage to structures other than thetarget tissues. For a primate, it is believed that the green porphyrinbegins to reach the retinal vasculature by about 7-15 seconds followingadministration. Typically, the green porphyrin persists for a period ofabout 5-15 minutes, depending on the dose given. Treatment within thefirst five minutes following administration of the green porphyrinshould generally be avoided to prevent undue damage to retinal vesselsstill containing relatively high concentrations of the green porphyrin.

Clinical examination and fundus photography typically reveal no colorchange immediately following photodynamic therapy, although a mildretinal whitening occurs in some cases after about 24 hours. Closure ofchoroidal neovascularization, however, is preferably confirmedhistologically by the observation of damage to endothelial cells.Vacuolated cytoplasm and abnormal nuclei can become apparent as early as1-2 hours following photodynamic therapy, with disruption of neovasculartissue typically becoming more apparent by about 24 hours after lighttreatment. Associated damage to the retinal pigment epithelium (RPE),pyknotic nuclei in the outer nuclear layer, and loss of photoreceptorsmay also be observed. However, the inner retina usually appearsrelatively undamaged, as shown by control studies using photodynamictherapy with BPD-MA on a normal retina and choroid showing no damage tolarge choroidal and retinal vessels.

Closure can usually be observed angiographically by about 40 seconds toa minute in the early frames by hypofluorescence in the treated areas.During the later angiographic frames, a corona of hyperfluorescencebegins to appear and then fills the treated area, possibly representingleakage from the adjacent choriocapillaris through damaged retinalpigment epithelium in the treated area. Large retinal vessels in thetreated area perfuse following photodynamic therapy, but tend todemonstrate late staining.

Minimal retinal damage is generally found on histopathologic correlationand is dependent on the fluence and the time interval after irradiationthat the green porphyrin is administered. Histopathologic examinationusually reveals vessel remnants in the area of choroidal neovasculartissue, but the retinal vessels typically appear normal. Further, thereis no indication of systemic toxicity, and cutaneous photosensitizationdoes not appear to develop.

As a result of the invention, photodynamic therapy can be used moreselectively, relying on the low intensity light exposure of greenporphyrins that have become localized within vascular tissue.Complications, such as hemorrhage, are not noted with the inventionmethod. Thus, photodynamic therapy with a green porphyrin appears tohave broad application to clinical ophthalmology in treating suchdiseases as age-related macular degeneration, neovascular glaucoma, andpersistent disc neovascularization in diabetic retinopathy.

The following examples are to illustrate but not to limit the invention.

EXAMPLE 1 Control of Experimental Choroidal Neovascularization Using PDTwith BPD-MA/LDL at Low Irradiance

Cynomolgus monkeys weighing 3-4 kg were anesthetized with anintramuscular injection of ketamine hydrochloride (20 mg/kg), diazepam(1 mg/kg), and atropine (0.125 mg/kg), with a supplement of 5-6 mg/kg ofketamine hydrochloride as needed. For topical anesthesia, proparacaine(0.5%) was used. The pupils were dilated with 2.5% phenylephrine and0.8% tropicamide.

Choroidal neovascularization was produced in the eyes of the monkeysusing a modification of the Ryan model, in which bums are placed in themacula, causing breaks in Bruch's membrane, with a Coherent Argon DyeLaser #920, Coherent Medical Laser, Palo Alto, Calif. (Ohkuma, H. et al.Arch. Ophthalmol. (1983) 101: 1102-1110; Ryan, S. J. Arch Ophthalmol(1982) 100:1804-1809). Initially, a power of 300-700 mW for 0.1 secondswas used to form spots of about 100 μm, but improved rates ofneovascularization were obtained with 50 μspots formed using a power ofabout 300-450 mW for 0.1 second.

The resulting choroidal neovascularizations were observed by (1) fundusphotography (using a Canon Fundus CF-60Z camera, Lake Success, LongIsland, N.Y.); (2) by fluorescein angiography (for example, by usingabout 0.1 ml/kg body weight of 10% sodium fluorescein via saphenous veininjection); and (3) histologic examination by light and electronmicroscopy.

Immediately before use, BPD-MA was dissolved in dimethyl sulfoxide(Aldrich Chemical Co., Inc., Milwaukee, Wis.) at a concentration ofabout 4 mg/ml. Dulbeccos phosphate buffered salt solution (Meditech,Washington, D.C.) was then added to the stock to achieve a final BPDconcentration of 0.8 mg/ml. Human low-density-lipoprotein (LDL) preparedfrom fresh frozen plasma was added at a ratio of 1:2.5 mg BPD-MA:LDL.The green porphyrin dye and dye solutions were protected from light atall times. After mixing, the dye preparation was incubated at 37° for 30minutes prior to intravenous injection. The monkeys were then injectedintravenously via a leg vein with 1-2 mg/kg of the BPD-MA complexed withLDL over a five-minute period, followed by a flush of 3-5 cc of normalsaline.

Following this intravenous injection, the eyes of the monkeys wereirradiated with 692 nm of light from an argon/dye laser (Coherent 920Coherent Medical Laser, Palo Alto, Calif.), using a Coherent LDS-20 slitlamp. The standard fiber was coupled to larger 400 μm silica opticalfiber (Coherent Medical Laser, Pal Alto, Calif.) to allow largertreatment spots as desired. Seventeen (17) areas of choroidalneovascularization were treated using a 1250 μm spot. Treatment spotsizes were confirmed at the treatment plane using a Dial calipermicrometer. Some areas of choroidal neovascularization were treated withseveral adjacent treatment spots to treat the whole area of choroidalneovascularization. One large choroidal neovascular membrane was treatedwith photodynamic therapy to the nasal half only.

The photodynamic irradiation treatments were carried out with a planofundus contact lens (OGFA, Ocular Instruments, Inc., Bellvue, Mass.).Power was verified at the cornea by a power meter (Coherent Fieldmaster,Coherent, Auborn, Calif.). The fluence at each treatment spot was 50,75, 100 or 150 Joules/cm². Initially, the irradiance was set at 150mW/cm² to avoid any thermal effect but, as the experiment proceeded, theirradiance was increased to 300 mW/cm² or 600 mW/cm2 to reduce thetreatment duration time. The time interval between injection of thegreen porphyrin dye and the treatment irradiating step ranged from about1 to about 81 minutes.

A number of different combinations of parameter values, were studied andare summarized below in Table 1: TABLE 1 IRRADIANCE AT 150 mW/cm² Numberof Duration of Time after CNV Dye dose Fluence Treatment InjectionClosure by Treated (mg/kg) (J/cm²) (mins) (mins) Angiography 2 2 50 5.618, 38 2/2 1 2 75 8.3 81 1/1 1 2 100 11.2 22 1/1 2 1 50 5.6  5, 30 0/2 31 100 11.2 1, 2 and 5 3/3 4 1 150 16.6 14-43 3/4

“Dye only” controls, which were exposed to dye but not to laser light,were in the areas of normal retina/choroid. Areas of choroidalneovascularization were angiographically and histologically. “Lightonly” controls were not performed, since ances used for photodynamictherapy were well below the levels used for clinical laser ulation. (Ina related experiment, a minimally detectable lesion using “light-only”an irradiance of 37 W/cm², about 100 times the light levels used forphotodynamic therapy.)

Following photodynamic therapy, the monkeys were returned to an animalcare facility. No attempt was made to occlude the animals'eyes, but theroom in which they were as darkened overnight.

The condition of the choroidal neovasculature was followed by fundusphotography, fluorescein angiography, and histologic examination. Inparticular, the eyes of the were examined by fluorescein angiographyacutely and at 24 hours after the photodynamic therapy was given. Insome cases, follow-up by fluorescein angiography was performed at 48hours and at one week, until the eyes were harvested and the animalskilled at the following time points: acutely, at 24 hours, 48 hours, and8 days following photodynamic therapy. Animals were sacrificed with anintravenous injection of 25 mg/mg Nembutal.

To perform the histologic examination, all eyes were enucleated underdeep anesthesia and fixed overnight in modified Karnovsky's fixative,and then transferred to 0.1 M phosphate buffer, pH 7.2 at 4° C. Bothlight microscopy and electron microscopy were used for these studies.For light microscopy, tissue samples were dehydrated, embedded in eponand serially sectioned at one micron. The sections were stained withtolnizin blue and examined with an Olympus photomicroscope. For electronmicroscopy, tissue samples were post-fixed in 2% osmium tetroxide anddehydrated in ethanol. Sections were stained with uranyl acetate inmethanol, stained with Sato's lead stain, and examined with a Philips#CM 10 transmission electron microscope.

Using the low irradiance level of 150 mW/cm² to minimize any thermalcomponent of the treatment, green porphyrin doses of 1-2 mg/kg ofBPD-MA/LDL, and fluences of 50-150 Joules/cm², choroidalneovascularization was effectively closed. Using the higher 2 mg/kg doseeffectively closed choroidal neovascularizations at even the lowest 50Joules/cm² fluence. When the green porphyrin dose was decreased to thedecrease the damage to surrounding tissues to 1 mg/kg of BPD-MA/LDL, thefluence required to effectively close choroidal neovascular tissueincreased to 100 Joules/cm². At 100 and 150 Joules/cm², the treatedchoroidal neovascular tissue was angiographically closed, as shown byhypofluorescence in the area of treatment.

Prior to photodynamic therapy, the areas of choroidal neovascularizationexhibited a gray sub-retinal elevation that leaked profusely onfluorescein angiography. There was no apparent color change in thetreated areas either during or imrnediately after photodynamictreatment. However, 24 hours after the irradiating step, there was mildretinal whitening in the treated areas.

Further fluorescein angiography showed hypofluorescence in the treatedareas, with no apparent filling of the associated neovascular tissues.Retinal vessels within the treated areas were perfused, but stainedlater. A hyperfluorescent rim at the border of the treated area wasapparent in the later frames of the angiograph, and the rim thenprogressed to fill the treated area. Although mild staining of retinalvessels was noted angiographically, no complications, such as serousretinal detachment or hemorrhage, were noted.

On histopathologic examination of the 2 mg/kg dose samples, there wasmarked disruption of the treated choroidal neovascular tissue withdisrupted endothelial cells. The choriocapillaris was also occluded.Although large choroidal vessels were unaffected, extravasated red bloodcells were noted in the choroid. Retinal pigment epithelium (RPE) damagewas noted as well with vacuolated cells, with the outer nuclear layerdemonstrating pyknotic nuclei and disrupted architecture. No histologicabnormality of the retinal vessels was seen.

Histopathologic examination of the 1 mg/kg dose samples showed damage toendothelial cells in the choroidal neovascular tissue, with abnormalnuclei and disrupted cytoplasm in the endothelial cells. The lumens ofthe vessels in the choroidal neovascular tissue were occluded by fibrinacutely and were closed by 24 hours after treatment. Closure of thechoriocapillaris was also noted. At 24 hours, the retinal pigmentepithelium (RPE) appeared abnormal with vacuolated cytoplasm. Pyknoticnuclei in the inner and outer layer indicated damage secondary to thelaser injury used to induce the neovascularization in this model.Retinal vessels appeared to be undamaged.

Choroidal neovascular tissue that was treated and followed for eightdays showed persistent closure, as shown by hypofluorescence in theearly frames of the angiogram. Histologically, the treated areasdemonstrated degraded vessel lumens empty of debris. Thechoriocapillaris was sparse but patent in the treated area. In contrast,areas of choroidal neovascularization not treated by photodynamictherapy demonstrated branching capillaries between Bruch's membrane andthe outer retina.

No adverse effects of photodynamic therapy with the green porphyrin werenoted. There was no associated serous retinal detachment, retinal orsub-retinal hemorrhage, or post-treatment inflammation. Further, noadverse systemic effects of the dye administration were noted. However,the low irradiance forced treatment times to be long—about 16.6 minutesto yield 150 Joules/cm².

EXAMPLE 2 Control of Experimental Choroidal Neovascularization Using PDTwith BPD-MA/LDL at Higher Irradiances

To make clinical treatments shorter, additional experiments wereperformed using higher irradiance values. Experience with higherirradiance indicated that no thermal damage would take place withirradiances as high as 1800 mW/cm². Moulton et al., “Response of Retinaland Choroidal Vessels to Photodynamic Therapy Using BenzoporphyrinDerivative Monoacid”, IOVS 34, 1169 (1993), Abstract 2294-58. Therefore,irradiances of 300 mW/cm² and 600 mW/cm² were also used to treatchoroidal neovascular tissue in accordance with the procedures describedin Example 1. The results showed that shortened treatment timeseffectively closed the choroidal neovascular tissue, as indicated belowin Table 2. TABLE 2 IRRADIANCE OVER 150 mW/cm² Duration Number ofClosure of Dye Treat- Time after by CNV does Fluence Irradiance mentInjection Angio- Treated (mg/kg) (J/cm²) (mW/cm²) (mins) (mins) graphy 21 150 300 8.3  5, 53 2/2 2 1 150 600 4.7 22, 69 2/2

Occlusion of the choroidal neovascular tissue and subjacentchoriocapillaris was observed, as well as damage to the retinal pigmentepithelium and outer retina.

EXAMPLE 3 Control of Experimental Choroidal Neovascularization Using PDTwith BPD-MA Liposomes

The following experiment of photodynamic therapy using a liposomalpreparation of BPD-MA was conducted to determine the optimal timeinterval after intravenous injection as a bolus of the BPD-MA over about20 seconds, followed by a 3-5 cc saline flush, to begin the irradiatingstep. Choroidal neovascularization in cynomolgus monkeys was treated todemonstrate efficacy of the photodynamic therapy. Normal choroid tissuewas treated to assess relative damage to adjacent tissues.

The monkeys were initially injected with a green porphyrin dose of 1mg/kg. At predetermined time intervals following this injection, theeyes of the monkeys were irradiated with an irradiance of 600 mW/cm²,and a fluence of 150 J/cm². The irradiating light was from an argon/dyelaser (Coherent 920 Coherent Medical Laser, Palo Alto, Calif.) equippedwith a 200 micron fiber adapted through a LaserLink (Coherent MedicalLaser) and a split lamp delivery system (Coherent). Other than thesedifferences, the eye membranes were treated in the same manner asdescribed in Example 1. All areas of treated choroidal neovasculaturefor all time points after the liposomal BPD-MA injection showedwhitening of the retina and early hypofluorescense on fluoresceinangiography when measured one week after treatment. On histology, therewas evidence of partial closure of choroidal neovasculature at the earlytime points, no effect at mid-time points, and more effective closure atlate irradiation time points, e.g., at 80 and 100 minutes.

The normal choroid treated with the same parameters showed whitening ofthe retina, early hypofluorescence at all time points, and histologicevidence of choriocapillaris (c-c) accompanied by damage to the choroidand retina, particularly at early time points.

EXAMPLE 4 Using PDT with BPD-MA Liposomes at Lower Green Porphyrin Doses

Using the general procedure of Example 1, additional experiments wereperformed using the intravenous injection of liposomal BPD-MA at dosesof 0.25, 0.5 and 1 mg/kg. Photodynamic therapy was performed with anirradiance of 600 mW/cm², a fluence of 150 J/cm², and a treatmentduration of four minutes, nine seconds.

The effects of treatment were assessed by fundus photography andfluorescein angiography, and then confirmed by light and electronmicroscopy. Photodynamic therapy of normal choroid tissue demonstratedthe effect on adjacent structures, such as the retina, while thetreatment of choroid neovascular tissue demonstrated efficacy.

Table 3 below describes the lesions produced on normal choroids byadministration of 0.5 mg/kg BPD-MA at time points ranging from 5 to 60minutes: TABLE 3 0.5 mg/kg, NORMAL CHOROID Time after injection (min)Fluorescein Angiography Histology 5 Hypofluorescence c—c and largechoroidal vessel closure; outer and inner retina damage 20Hypofluorescence; retinal cc closure; damage to vessels - normal outerretina 40 Mild early cc open (not center of hypofluorescence lesion);outer retina damage 60 Early hypofluorescence; cc closed; outer retinaless than the 20-minute damage; inner retina lesion described abovefairly good.

When 0.5 mg/kg BPD-MA was also used to treat choroidal neovasculatureunder the same conditions marked hypofluorescence corresponding toclosure of choroid neovasculature was exhibited in areas irradiated attimes of 5, 20 and 40 minutes after injection. When 50 minutes afterinjection were allowed to elapse before photodynamic irradiation wasbegun, there was less hypofluorescence and presumably less effectiveclosure.

The study was then repeated with the green porphyrin dose decreased to0.25 mg/kg. Table 4 describes the lesions produced on normal choroids bytreatments with 0.25 mg/kg, 600 mW/cm², and 150 J/cm² at time pointsranging from 5 to 60 minutes: TABLE 4 0.25 mg/kg, NORMAL CHOROID Timeafter injection Fluorescein (min) Angiography Histology 10 Earlyhypofluorescence c—c closure; choroidal vessel - normal; RPE damaged;retinal vessels - normal; mild damage to outer retina 20 Earlyhypofluorescence Same as 10-minute lesion above 40 Faint early Patchy ccclosure; less damage to hypofluorescence; late RPE and outer retinastaining 60 Not demonstrated No effect on cc; mild vacuolization of RPE

When the above study was repeated using the same green porphyrin dose of0.25 mg/kg and irradiance of 600 mW/cm², but with a reduced fluence of100 J/cm², the same angiographic and histologic pattern was exhibited asdescribed above. However, cc was open in the 40-minute lesion.

In the last portion of these experiments, a green porphyrin dose of 0.25mg/kg was used to treat expermental choroidal neovascularization with anirradiance of 600 mW/cm² and a fluence of 150 J/cm² at elapsed timepoints ranging from 5 to 100 minutes. This combination of ffective ccclosure with only minimal damage to the outer retina. The results areshown in Table 5 below: TABLE 5 0.25 mg/kg, PDT over CNV Time afterinjection (min) Fluorescein Angiography Histology 5 Earlyhypofluorescence Partially closed CNV; c—c closed; damage to innerretina 20 Early hypofluorescence; CNV-open vessel, fibrin and clots;less than the 5-minute inner retina looks fine lesion 30 Somehypofluorescense Minimal effect on CNV next to CNV 40 Hypofluorescence;Minimal effect on CNV questionable change compared to previous reaction60 Hypofluorescence Minimal effect on CNV 80 Hypofluorescence Partialclosure of CNV; retina over CNV looks intact 100 Hypofluorescence CNVpartially closed

Thus, fluorescein angiography and histopathology in the above series ofexperiments demonstrated early hypofluorescence at early time points.Further, the hisopathology study showed partial CNV closure at all timepoints after injection using 80 and 100 minutes as the post-injectioninterval before the irradiating treatment.

In summary, acceptable destruction of choroidal neovascular tissue atall tested doses of BPD-MA was shown by fluorescein angiography andhistology. However, the lower doses appeared to increase selectivity, asassessed by treatment of a normal choroid. Effective choriocapillarisclosure in normal choroids with minimal retinal damage was produced byirradiating about 10 minutes, 20 seconds after injection of the greenporphyrin at a dose of 0.25 mg/kg. By adjusting the dose, the time ofirradiation after green porphyrin injection, and fluence, one canimprove even further the selectivity of the green porphyrin. However,the liposomal preparation of BPD-MA was clearly demonstrated to be apotent photosensitizer.

EXAMPLE 5 Additional Data Using Liposomal BPD

Using the techniques of Example 1-4, a total of 61 areas of experimentalCNV in 9 monkeys were treated with PDT using BPD-MA. Effective CNVclosure was demonstrated by fluorescein angiography at all tested dyedoses: 1, 0.5, 0.375, and 0.25 mg/kg. The lower the dose, the shorterthe time interval after dye injection in which laser irradiationproduced CNV closure.

The fundus appearance was unchanged immediately after treatment, andonly slight deep retinal whitening corresponding to the laserirradiation spot appears 24 hours later. CNV closure was determinedangiographically at 24 hours by early hypofluorescence corresponding tothe treated area. As the angiogram progressed most lesions demonstratedstaining starting at the periphery of the lesion.

Table 6 summarizes the effect of PDT on CNV, using different dye dosesand variable treatment times after dye injection. PDT using a dye doseof 1 mg/kg was performed over 7 membranes in 1 monkey. Laser irradiationwas performed at each of the following times after dye injection: 5, 20,40, 60, 80, 100 and 120 minutes. CNV closure was induced in all lesionswhen irradiation was performed 5-100 minutes after dye injection. TABLE6 Angiographic Closure of CNV Dye Dose No. Lesions Time (min) of R_(x)after CNV mg/kg CNV dye injection Closure 1 7  5-100 6/7 >100  0/1 0.511 <60 7/8  60-100 0/3 0.375 29 <50 16/18  50-100  3/11 0.25 14 <20 2/220-40 2/4  40-100 0/8

PDT using dye dose of 0.5 mg/kg was performed on 11 membranes in 2monkeys, with laser irradiation at 10, 20, 30, 40, 50, 60, 80, 100minutes after dye injection. PDT effect was assessed 24 hours aftertreatment. CNV closure was induced in 7/11 membranes, that wereirradiated at 10, 20, 30, 40 and 50 minutes after dye injection. Only1/2 membranes irradiated at 50 minutes after dye injection showedangiographic closure. The treatments performed 60 minutes and more afterdye injection showed no angiographic closure of the membranes.

29 areas of CNV in 5 monkeys were treated with PDT using BPD-MA at doseof 0.375 mg/kg. All treated CNV membranes were assessed angiographicallyat 24 hours. As indicated in Table 6, 7/8 CNV irradiated within 50minutes after injection demonstrated angiographic closure. Only 3/11membranes irradiated more than 50 minutes after dye injectiondemonstrated angiographic closure.

A dye dose of 0.25 mg/kg was found to be the threshold dose for PDTusing a light dose of 150 J/cm² and 600 mW/cm² CNV closure wasdemonstrated in 2/2 membranes that were irradiated within 20 minutesafter dye injection. Only 2/4 CNV irradiated 20-40 minutes after dyeinjection showed closure. No effect was demonstrated in the CNV thatwere irradiated more than 40 minutes after dye injection.

Histologic confirmation of CNV closure was evident at all tested dyedoses: 1, 0.5, 0.375, and 0.25 mg/kg.

On light microscopy the closed CNV showed vessels packed with red bloodcells (RBCs), occasional extravasated RBCs and pockets of fibrin withinthe tissue as well as in the subretinal space. Most of the stromal cellsappeared undamaged.

On electron microscopy the closed vessels appeared packed with RBCs andplatelets. The endothelial cells were missing or severely damaged.Extravasated RBCs and occasional white blood cells (WBCs) were foundnear the vessel remnants. At 0.25 mg/kg the vessels were packed withRBCs but the endothelial cells seemed to be surviving the treatment.

Treatment Selectivity

Treatment selectivity was investigated by performing PDT in normalretina/choroid using the same dye doses and time points of laserirradiation after dye injection. In most cases the closure of thechoriocapillaris in normal choroid followed a similar time course as theclosure of CNV. When PDT was performed using dye doses of 0.5, 0.375,0.25 mg/kg, the retinal structure was well preserved. In none of thecases were retinal detachment or hemorrhage observed. reducing the dyedose resulted in more selective closure of the choriocapillaris withminimal damage to the adjacent tissues. RPE cells were typically damagedat all dye doses.

The assessment of the damage to the retina and choroid was gradedaccording to the histologic findings for the retina/choroid at differentlevels, as follows:

-   Grade 1: RPE only or RPE+slight photoreceptor changes+occasional    pyknosis in the ONL; with or without choriocapillaris (c-c) closure;-   Grade 2: Choriocapillaris closure+RPE+photoreceptors+10-20% pyknosis    in the ONL;-   Grade 3: C-c closure+RPE+photoreceptors+ONL pyknosis>50%;-   Grade 4: C-c closure+RPE+photoreceptors+ONL pyknosis>50%;-   Grade 5: C-c closure+RPE+photoreceptors+ONL pyknosis>50%+choroidal    vessel damage or retinal vessel or inner retinal damage;

A total of 38 PDT spots were placed in normal retina/choroid. Thetreatment parameters and the degree of effect are summarized in Table 7.TABLE 7 PDT effect on normal retina/choroid Time (min) No. of lesionsper of R_(x) after histologic grading Dye Dose dye injection No. ofLesions 1 2 3 4 5 1 mg/kg  <60* 2 2  60-100* 3 3 0.5 mg/kg <20 1 1 20-603 3 0.375 mg/kg <20 3 1 1 1 20-50 9 2 4 2 1  50-100 11 1 5 5 0.25 mg/kg<20 1 1 20-40 1 1 40-60 2 2*The lesions irradiated at 40 and 120 minutes were not identifiedhistologically.

PDT using a dye dose of 1 mg/kg led to damage of both inner and outerretina. The early treatments (5 and 20 minutes after dye injection)demonstrated grade 5 effect with damage to the inner retina, and lesionsinduced 60 minutes and more after dye injection showed a grade 4 effect.

At 0.5 mg/kg, only the lesion irradiated 5 minutes after dye injectiondemonstrated damage to the inner retina (grade 5). Lesions irradiated at20 minutes and later did not affect the inner retina, but showedpyknosis in the outer nuclear layer (ONL), vacuolization anddisorganization of the photoreceptors'inner and outer segments, anddamage to the RPE (grade 4).

At 0.375 mg/kg, 2/3 lesions irradiated 10 minutes after dye injectionshowed ngestion of the small retinal vessels, but the inner nuclearlayer (INL) was preserved. applied 20 minutes and later after dyeinjection showed some pyknosis in the ONL, cuolization anddisorientation of the photoreceptors'inner and outer segments, and tothe RPE. Most lesions demonstrated damage of grade 1 or 2 or 3, withsome lesions rated grade 4 damage.

0.25 mg/kg was found to be a threshold dose for induction ofchoriocapillaris closure. This was achieved with almost no effect on theoverlying retina. There was mild damage to some RPE cells, minimalswelling of photoreceptors, and a few pyknotic nuclei in the ONL.

“Dye only” control areas of normal retina/choroid showed no effect byfluorescein angiography or histologic examination.

1-20. (canceled)
 21. A method of treating unwanted choroidalneovasculature in a shortened treatment time, the method comprising thesteps of: administering to a primate subject in need of such treatmentan amount of a porphyrin dye sufficient to permit an effective amount tolocalize in the neovasculature; and irradiating the neovasculature withlight having an irradiance in the range from at least about 300 mW/cm²to about 900 mW/cm², the light being absorbed by the porphyrin dye so asto occlude the neovasculature.
 22. The method of claim 21, wherein theporphyrin is green porphyrin.
 23. The method of claim 25, wherein thelight has a fluence of 50 Joules/cm².
 24. The method of claim 21,wherein the primate subject has age-related macular degeneration andocclusion of the neovasculature ameliorates symptoms of the age-relatedmacular degeneration.
 25. The method of claim 22, wherein the primatesubject has a disorder selected from the group consisting of age relatedmacular degeneration, ocular histoplasmosis syndrome and myopia, whereinocclusion of the neovasculature ameliorates the disorder.
 26. A methodof treating age-related macular degeneration in a primate subject havingunwanted choroidal neovasculature in a shortened treatment time, themethod comprising the steps of: administering to the primate subject anamount of a green porphyrin dye sufficient to permit an effective amountto localize in the neovasculature; and irradiating the neovasculaturewith light having an irradiance in the range from at least about 300mW/cm² to about 900 mW/cm², the light being absorbed by the porphyrindye so as to occlude the neovasculature.
 27. The method of claim 26,wherein the light has a fluence of 50 Joules/cm².